SbbHLH85, a bHLH Member, Modulates Resilience to Salt Stress By Regulating Root Hair Growth in Sweet Sorghum

Introduction: bHLH family proteins play an important role in plant stress response. However, the molecular mechanism regulating salt response of bHLH is largely unknown. Materials and Methods: Here, we investigated the function and regulating mechanism of the sweet sorghum SbbHLH85 during salt stress. Results: Results show that SbbHLH85, different from its homologs in other species, is a new atypical bHLH transcription factor and is a key gene for root development in sweet sorghum. Knockout of SbbHLH85 in sorghum by CRISPR-Cas9 results in the inhibition of root development. Overexpression of SbbHLH85 resulted in signicantly increased number and length of root hairs by participating in ABA and auxin signaling pathways, which can increase the absorption of Na + . While SbbHLH85 plays a negative regulatory role in salt tolerance of sorghum. Through screening yeast two-hybrid library, we identied a potential interaction partner of SbbHLH85 that is phosphate transporter chaperone PHF1, which modulates the distribution of phosphate. Both yeast two-hybrid and BiFC experiments conrmed interaction between SbbHLH85 and PHF1. Discussion: Based on these results, we suggest that the increase of Na + content and the decrease of Pi content result in the salt sensitivity of transgenic sorghum.


Introduction
The problem of soil salinization exists widely in the world. Salt stress caused by soil salinization affects the growth, development and harvest yield of plants, and in serious cases salt stress even leads to plant death (Asano et al., 2012;Sui et al., 2017). The most cost-effective way to utilize salinized soil is to develop salt-tolerant crop varieties based on well-established knowledge of molecular mechanisms for plant salt resistance, therefore promoting agricultural production. Sweet sorghum (Sorghum bicolor (L.) Moench) is a crop with high sugar content and high biomass yield, which has the laudatory name of second-generation sugarcane (Sui et al., 2015). In today's world, it is an important food, feed and energy crop. In addition to high biomass, sweet sorghum also has tolerance to various abiotic stresses, especially to salt (Schnippenkoetter et al., 2017). Many genes have been found to play an important role in regulating salt tolerance in sweet sorghum (Song et al., 2020;Wang et al., 2014b;Yang et al., 2018;Zheng et al., 2011). It is believed that the outcomes from studying characteristics of these genes can be applied to improve other crops for salt tolerance, which also is of great signi cance for understanding plant growth and development in saline alkali environment.
Salt stress increases the level of sodium ion (Na + ) and potassium ion (K + ), thus reducing the level of nutrient elements (e.g., N P) and causing the imbalance of nutrient distribution in plants (Colla et  Phosphorus transfer between different tissues and subcellular organelles in plants are facilitated with a series of transport proteins having phosphorus transfer activity (Deng et al., 2017). The amount of phosphotransporters on the plasma membrane directly determines the absorption and distribution e ciency of phosphorus in plants (Mudge et al., 2002). PHT1 encodes a phosphoric acid (Pi) transporter, which is regulated by the plant-speci c phosphate transport chaperone PHF1 during its transport to plasma membrane, and plays a fundamental role in the acquisition and reactivation of Pi ( . When the bHLH transcription factor AtNIG1 was knocked out, the resulting mutant became more sensitive to salt stress in Arabidopsis. AtNIG1 regulates salt tolerance by binding speci cally to the E-box sequences in the promoter regions of many salt stress-related genes (Kim and Kim, 2006).
Studies have shown that the plant bHLH transcription factors are also involved in root hair formation (Gajewska et al., 2018;Yan et al., 2014). Root hair is a tubular protrusion formed by extension of root speci c epidermal cells, which contains enzymes and nutrient transporters involved in nutrient absorption (Menand et al., 2007;Wei and Li, 2018). Root hairs increase the contact area between plants and soil and determine the e ciency of root absorption of water and nutrients (Krasilnikoff et al., 2003). The Arabidopsis bHLH transcription factors GLABRA3 (GL3) and ENHNACER OF GLABRA3 (EGL3) have redundant functions. The number of root hairs in the single mutants of these two genes increased slightly, whereas the double mutants increased root hair number signi cantly (Bernhardt et al., 2005). The polar growth of root hair is initially triggered by RHD6/RSL1 of the bHLH family, and then its elongation is activated by the expression of RSL4/RSL2 (Vijayakumar et al., 2016). RSL4 is essential for root hair elongation in Arabidopsis, which controls the nal root hair cell size (Zhu et al., 2020). RSL2 affects ROS production and root hair growth (Rymen et al., 2017).
In this study, we rst cloned a bHLH gene, SbbHLH85 in sweet sorghum, which is induced by salt stress and ABA. Some reports have shown involvement of the bHLH transcription factor in salt stress response and root hair formation. In order to investigate the molecular mechanism on how SbbHLH85 regulates root hair development in response to salt stress in sweet sorghum, we rst overexpressed SbbHLH85 in sweet sorghum and Arabidopsis. Results showed that different from its homologs in other species, the sweet sorghum SbbHLH85, is a new atypical bHLH transcription factor and is a key gene for root development in sweet sorghum. Knockout of SbbHLH85 in sorghum by CRISPR-Cas9 results in the inhibition of root development. Overexpression of SbbHLH85 in sorghum and Arabidopsis signi cantly increase the number and length of root hairs. While salt resistance was signi cantly lower in those overexpression lines. Overexpression of SbbHLH85 can in uence the expression of the genes involved in ABA and auxin signal transduction (PYL and PIN3), peroxidase (PERs), and root hair development and receptor-like proteins (RLKs). In addition, SbbHLH85 interacts directly with SbPHF1, a phosphate transporter chaperone protein in sweet sorghum, affecting the transport of Pi. Based on these results, we suggest that SbbHLH85 participates in regulation of ABA and auxin signal transduction pathways and distribution of nutrients to affect the development of root hairs, thus affecting the absorption of Na + and the content of ROS, and mediating plant salt response.

Plant materials and growth conditions
In this study, wild type Arabidopsis was used as a control. The WT and mutant seeds were evenly seeded on 1/2 MS medium with corresponding antibiotics. The culture dish was placed at low temperature for 3 days and cultured in the tissue culture room (22℃-16 h light/18℃-8 h dark), and the screened positive seedlings were transferred to nutrient soil for further cultivation until the seeds mature. The harvested seeds were seeded with the same operation in 1/2 MS medium with salt, and the phenotype of the plants was observed.
Sweet sorghum (Sorghum bicolor (L.) Moench) cultivar M-81E was used in this experiment. The seeds of sweet sorghum were cultured in sand. Tap water was irrigated before emergence and 1/2 Hoagland nutrient solution was poured every day after emergence. When the seedlings grew to three leaves and one heart, Hoagland nutrient solution containing 0, 50, 100, 150 and 200 mM NaCl was applied to sweet sorghum under salt stress.

Cloning, bioinformatics and expression analysis of SbbHLH85
The SbbHLH85 CDS sequence is obtained by comparing the AtRSL2 sequence on the Ensembl website (http://ensembl.gramene.org/). Online website NCBI (https://www.ncbi.nlm.nih.gov/), SMART (http://smart.embl-heidelberg.de/) and software MEGA6 for nucleic acid and protein sequence analysis and the construction of evolutionary trees.
Root tissue of sweet sorghum was used for cloning and expression of SbbHLH85. Samples were immediately frozen in liquid nitrogen and stored -80℃ before analysis. Three biological replicates were performed for RT-qPCR. Sweet sorghum internal reference gene Sbactin-1 as a control, the primers are listed in supplementary table 1.

Subcellular localization of SbbHLH85 protein
Using KpnI and BamHI sites to insert cloned SbbHLH85 CDS into pCAMBIA1300-35S-sGFP vectors to produces 35 S SbbHLH85-GFP constructs. It was transferred into Agrobacterium tumefaciens GV3101 and used to infect tobacco epidermal cells. GFP signal was observed by two-photon laser scanning confocal microscope (TCS S8MP, leica, germany). 35S: GFP transgenic tobacco was used as a localization control for expression in cytoplasm/nucleus. The primers are listed in supplementary table 1.

Generation of transgenic plants
To produce overexpressed SbbHLH85 of Arabidopsis, SbbHLH85 genes were linked into pROKII vectors with XbaI and KpnI and transferred to Agrobacterium tumefaciens GV3101 to obtain SbbHLH85 overexpressed plants by infecting WT in orescence. The transgenic Arabidopsis plants were screened with kanamycin (50 g/mL) and veri ed by RT-PCR. Insert the full-length cDNA of SbbHLH85 into the pMWB110 vector through BamHI and KpnI sites to obtain pMWB110-SbbHLH85 vector. pMWB110-SbbHLH85 vector was introduced into sweet sorghum by Agrobacterium-mediated transformation. PCR, herbicide (glufosinate) spraying and bar rapid detection kit were used to detect transgenic plants. The primers used are listed in supplementary table 1.
Quanti cation of biomass, MDA content, Na + and K + content For the measurement of biomass, we rst take the whole plant out of the pot, wash it, weigh it and record it as fresh weight. Then they were dried in the oven for 7 days and then weighed again as dry weight. Fresh weight and dry weight of each treatment were measured 5 times (Song et al., 2019). The MDA contents were determined as described by Ma(Ma et al., 2013). As follows: the leaves of 0.2 g of each line were weighed, 5 ml 0.1% trichloroacetic acid (TCA) was added for grinding. Mix the homogenate with 5 ml 0.5% thiobarbituric acid (TBA), boil for 10 min and take it out, cool to room temperature, 3000 rpm/min centrifuge 15 min, absorb the supernatant and measure its volume. The absorbance of the solution at wavelength 532 nm and 600 nm was determined by UV spectrophotometer. Blank control was 0.5% TBA solution. For the determination of Na + and K + , the speci c steps are described by Song: each line treated with 0 and 100 mM NaCl. After 10 d, put 0.3 g roots in 5 ml ddH 2 O, boil 2 h, lter plant residue and volume to 10 ml. The content of Na + and K + of each treated lines was determined by ame spectrophotometer (Song et al., 2020).

DAB and NBT staining
When Arabidopsis seedlings growing for about a month, treated with 0 or 100 mM NaCl in 1/2 concentration Hoagland solution for 48 h. Before staining leaves of rosette leaves of WT and overexpressed plants with the same growth, put in DAB or NBT dye solution, immerse the dye in the leaves, placed in the dark for more than 12 hours. Then put in the bleach (3:1:1 ethanol: acetic acid: glycerol), boil in boiling water for 10-15 minutes for decolorization, observe the color change of the blade and take the image.

Root hair experiment
The homologous gene of SbbHLH85 is AtRSL2 and AtRSL4 in Arabidopsis. AtRSL2 is closely related to root hair development and elongation, and it also has a functional redundancy gene AtRSL4. So we selected WT, M-81E, overexpression (At-OX4 At-OX13, Sb-OX1, Sb-OX3, Sb-OX6, Sb-OX7), RSL2 mutant (rsl2-1 rsl2-3), RSL4 mutant (rsl4), and RSL2 and RSL4 double mutant (rsl2rsl4) as experimental subjects, the main root hair development and root hair elongation were tested. Each Arabidopsis seed was on demand in 1/2 MS medium. The sweet sorghum seeds are hydroponic. After 7 d in the lab, development and elongation of root hairs at the root tip 5 mm from the main root of each line were observed under electron microscope. The microscope was magni ed by 40 times.

RNA-seq assay
The roots of WT, overexpression, mutant and double mutant were collected and preserved in liquid nitrogen. RNA-seq and differential gene expression analysis were carried out by BMK. Transcriptome analysis of 24 samples was completed, and 156.37 Gb of clean data was obtained. Use the HISAT2 system to sequenced the clean reads of each sample with the designated reference genome, and the reads on was assembled by StringTie comparison.
After the comparison and analysis, the reads on the sample were assembled and quanti ed by StringTie comparison. Based on the comparison results, the gene expression was analyzed. StringTie uses FPKM (fragments per kilobase of script per million fragments mapped) as an index to measure the expression level of transcripts or genes. The differentially expressed genes were identi ed according to their expression levels in different samples, and through GO database, KOG database and KEGG database for function annotation and enrichment analysis. KEGG pathway analysis was carried out for the common differential genes in each comparison group, and the heat map clustering analysis was performed for the expression level of selected genes, which were all completed on the platform of BMKCloud.

RT-PCR validation of genes related to plant salt resistance and root hair development
We extracted RNA from root tissues of WT, At-OX13, rsl2-3 and rsl2rsl4 lines which had been growing for about one month, and then reverse them into cDNA and then conduct RT-PCR. The primers used are in supplementary table 2. In sweet sorghum, M-81E and Sb-OX1 were treated with 0 mm and 100 mM NaCl for 48 h. Then RNA was extracted and transformed into cDNA, RT-PCR was performed. The primers used are in supplementary table 3.

Yeast two-hybrid experiment
The CDs of SbbHLH85 were cloned into the bait vector pGBKT7 to obtain SbbHLH85-BD, and then transformed into yeast strain Y2HGold (Clontech). After detecting that the gene has no self-activating activity, we constructed a yeast two-hybrid library of sweet sorghum, and hybridized and screened according to the matching scheme described in Clontech's matchmaker tmgoldyeasttwo hybrid user's manual. After screening, the interaction between SbPHF1 and SbbHLH85 was veri ed by yeast two-hybrid experiment. The full-length coding sequence (CDS) of SbPHF1 was cloned into pGADT7 to obtain SbPHF1-AD, and SbbHLH85-BD and SbPHF1-AD were co-transformed into Y2HGold. Then the growth of the colony on the corresponding medium was observed. See supplementary table 1 for the primers used in yeast two-hybrid.

BiFC experiment
The CDs of SbbHLH85 and the N-terminal of pSPYNE-35S were fused to obtain SbbHLH85-N-YFP, and the CDs of SbPHF1 and the C-terminal of pSPYNE-35S were fused to obtain SbPHF1-C-YFP. The obtained plasmid was introduced into Agrobacterium tumefaciens (GV3101), and the method of transient transformation was used to in ltrate the tobacco. After 48 hours of normal culture, the uorescence was observed under a confocal laser scanning microscope (Olympus). Supplementary table 1 lists the primers of BiFC.

Statistical methods
The statistical results are described as mean ± standard deviation. Use the SPSS ver. 17.0 statistical software to analyze the data. One-way ANOVA was used as the designated package. Different letters indicate that there is a signi cant difference between the average (0.05) of the Duncan test.

Results
SbbHLH85 is a salt stress responsive gene As a preliminary characterization of the function of SbbHLH85, we studied its conserved domain, expression pattern and subcellular localization. First, the phylogenetic tree analysis based on protein sequence showed that SbbHLH85 was closely related to A0A2S3HF40 in Panicum hallii and A0A3L6RBM2 in Panicum miliaceum. SbbHLH85 shares 33.15% and 32.18% homology with the Arabidopsis genes AtRSL2 and AtRSL4, respectively ( Figure 1A). After analyzing the domain structure, we found that they all have a bHLH domain at the C end ( Figure 1B). The SbbHLH85 gene contains a 951-bp coding sequence that encodes 316 amino acids. Different from RSL2 and RSL4, the SbbHLH85 protein contains a bHLH domain between amino acids 238 and 287, which contains a Q5-A9-R13 motif harboring a glutamine at the fth position, alanine at the ninth position and arginine at the thirteenth position ( Figure 1C; Supplementary Figure 1). By measuring the relative abundance of SbbHLH85 in roots of M-81E under different salt stress, the expression pattern of SbbHLH85 was further studied. RT-PCR results showed that the expression of SbbHLH85 in sweet sorghum roots decreased with the increase of salt concentration, dropping to the lowest level at 100 mM NaCl ( Figure 1D). The subcellular distribution of SbbHLH85 was studied by fusing SbbHLH85 with the green uorescent protein (GFP). In the lower epidermal cells of tobacco, signals were detected in the nucleus ( Figure 1E). Therefore, SbbHLH85 is a bHLH transcription factor that is negatively induced by salt and localized in the nucleus.

SbbHLH85actively regulates the development of root hairs
To explore the effect of SbbHLH85 on root hair development, we generated SbbHLH85 overexpression lines At-OX4 and At-OX13 controlled by the CaMV 35S promoter in Arabidopsis (Supplementary Figure 2). For the knockout of SbbHLH85 in sorghum by CRISPR-Cas9 results in the inhibition of root development, we use the AtRSL2 mutants rsl2-1 and rsl2-3, and the AtRSL4 mutant rsl4 to test the function of SbbHLH85 on root development. All the Arabidopsis mutants were purchased from the TAIR website (https://www.arabidopsis.org/). The double mutant rsl2rsl4 was provided as a gift by Professor Hongwei Guo of Southern University of Science and Technology. Then, the complementing lines Crsl2-1, Crsl2-3, Crsl4, Crsl2rsl4 were created by expressing SbbHLH85 in rsl2-1, rsl2-3, rsl4 and rsl2rsl4, respectively. Figure 2A shows that the number and length of root hairs in the overexpression lines were the largest and the longest, whereas the control mutants rsl2-1, rsl2-3 and rsl4 had fewer and shorter root hairs. Further, there was no root hairs observed in the double mutant rsl2rsl4. Thus, SbbHLH85 is able to complement the root hair defect in the single and double mutants (Figure 2A-C).
To further explore the effect of SbbHLH85 on root hair development, we constructed SbbHLH85 overexpression lines controlled by CaMV 35S promoter in sorghum, and detected the relative expression of SbbHLH85 in each line by RT-PCR. Compared with the wild type, SbbHLH85 was highly expressed in Sb-OX1, Sb-OX3, Sb-OX6 and Sb-OX7 (Supplementary Figure 3). It was also found that SbbHLH85 overexpression increased the number and length of root hairs in sweet sorghum ( Figure 2D-F). We also attempted the gene-editing tool CRISPR-Cas9 to knockout SbbHLH85 in sweet sorghum. However, we repeatedly observed inhibition of root growth in the transgenic plants carrying the SbbHLH85-Cas9 vector (Supplementary Figure 4) and as a result, we was unable to acquire the knockout mutants of sorghum. These ndings suggest that SbbHLH85, different from RSL2 and RSL4 in Arabidopsis, is necessary for root development in sorghum.

SbbHLH85 negatively regulates salt stress response
To explore the role of SbbHLH85 in regulating salt stress response in plants, we tested each of the Arabidopsis overexpression lines. We studied the effects of salt stress on plant growth, including germination rate and seedling survival. For the germinating plants, we sowed seeds of each line on 1/2 Murashige and Skoog (MS) medium containing 0, 100 or 150 mM NaCl. It was found that overexpression plants grew more slowly than WT and the mutant lines on the salt medium ( Figure 3A). By measuring the germination rate and root length of each line, it was found that the germination rate and root length of overexpression plants were higher than that of WT and mutants no matter under the condition of non-stressed or salt stress ( Figure 3B, C). Under abiotic stress, plants are exposed to ion stress, oxidative stress and osmotic stress at the same time. Next, we compared the germination rate and root length of each lines under different stress conditions. The growth of seedlings in NaCl, LiCl and mannitol media showed different degrees of slow growth ( Figure 3D). Among them, NaCl treatment resulted in the worst growth, the shortest taproot length and the lowest germination rate ( Figure 3E, F). We also studied the salt tolerance of different lines at seedling stage. Salt stress reduced the biomass of each line, but the fresh weight and dry weight of mutant lines decreased less ( Figure 3G, H). The contents of MDA increased more in WT and overexpression plants, but less in mutant lines ( Figure 3I). Ion content analysis under salt stress showed that Na + content in overexpression lines were higher than that in WT control ( Figure 3J . Under salt stress, DAB and NBT staining of overexpression lines were the deepest, which indicated that overexpression of SbbHLH85 caused more ROS production in plants ( Figure 3L, M). We also transformed SbbHLH85 to the mutant lines to investigate if SbbHLH85 can rescue the salt sensitive phenotype. The results showed that the physiological indexes of the complemental lines Crsl2-1, Crsl2-3, Crsl4, and Crsl2rsl4 under salt stress recovered ( Figure 4). Overall, these ndings suggest that ectopic overexpression of SbbHLH85 in Arabidopsis may affect the homeostasis of Na + and K + , the content of ROS and the degree of membrane lipid peroxidation to improve salt tolerance.
To better understand the function of SbbHLH85 in salt stress, we further tested the salt tolerance of the SbbHLH85 overexpression lines in sweet sorghum. Under salt stress, the overexpression lines of sweet sorghum showed obvious weak growth, yellow leaves and even curly symptoms ( Figure 5A). Next, we studied the biological processes of salt stress. The biomass accumulation of overexpression lines was lower than that of M-81E ( Figure 5B-E). Under salt stress, the content of Na + in overexpression lines increased signi cantly and the content of K + decreased ( Figure 5F-H). Compared with the WT of M-81E, MDA content of overexpression lines increased signi cantly after salt treatment ( Figure 5I). This is consistent with the conclusion from Arabidopsis that SbbHLH85 participates in salt stress response.

Transcriptome analysis of plants with altered SbbHLH85 level
To reveal the molecular mechanism of SbbHLH85 regulating plant response to salt stress and root hair development, we analyzed the RNA-seq data of WT, overexpression, mutant and double mutant plants under control and NaCl treatment. In order to verify the accuracy of transcriptome, we selected 22 representative genes for RT-PCR veri cation, and found that the transcriptome data were in high quality ( Figure 6E). In the RNA-seq experiment, we used 48 h NaCl treatment and three biological replications. Under salt stress, there were 597 differentially expressed genes (DEGs) in WT, 244 DEGs in overexpression lines, 204 DGEs in single mutants and 278 DGEs in double mutants. We intersected these DGEs and got 156 DGEs ( Figure 6A). Then, we used hierarchical clustering and correlation analysis to analyze the expression patterns of 156 DEGs genes. GO (Gene Ontology Consortium) analysis showed that these differential genes were involved in biological process, molecular function and cellular component, among which they are mainly involved in biological process ( Figure 6B). KEGG (Kyoto Encyclopedia of Genes and Genomes) and KOG (Cluster of Orthologous Groups of proteins) analysis showed that these differential genes were mainly related to phenylalanine metabolism, hormone signal transduction and secondary metabolite metabolism ( Figure 6C, D). After further analysis of the genes in these pathways, we found that they are mainly involved in auxin signaling pathway (AtPIN3, AtSAUR50), ABA signaling pathway (AtPYL6), and also involved in the production of peroxidase (PER) (AtPRX33, AtPRX37, AtRCI3, AT4G08780) and receptor like kinases (RLK) (AT4G00970, AT4G04570) ( Figure 6F) and development of root hairs ( Figure 6G). This is consistent with our previous conclusion that SbbHLH85 participates in salt stress and affects the development of root hair.
For the SbbHLH85 has a unique Q5-A9-R13 motif, it can't directly bind to the promoter of the downstream genes. In order to further study the molecular mechanism of SbbHLH85 regulating salt response and root hair development of sweet sorghum, all we can do is to identify all sweet sorghum genes homologous to the Arabidopsis ones. These genes are annotated as auxin signal, ABA signal, root hair development, PER and RLK. This study identi ed one ABA signal transduction gene (SbPYL4), one auxin signal transduction gene (SbPIN3), three root hair development related genes (SbRSH2, SbRSL4, SbRHL1), four PER genes (SbPER3, SbGLO1, SbPER4, SbPER35) and three RLK related genes (SbRLK1, SbRLK2, SbRLK8) in sweet sorghum ( Figure 6H). This is consistent with the previous results, indicating that SbbHLH85 is involved in root hair development and salt tolerance of sweet sorghum.

Interaction of SbbHLH85 with SbPHF1
Increasing lines of evidence show that the bHLH proteins act by forming protein complexes with other interacting proteins (Abe et al., 2003;Oh et al., 2007). In order to nd the potential chaperone of the bHLH85 protein, we used yeast two-hybrid system to screen for interactors. Firstly, the BD domain of pGBKT7 and bHLH85 was fused as bait. After we proved that bHLH85 had no self-activating activity ( Figure 7A), the cDNA library containing the prey protein insert fused with GAL4-AD was used to cotransform the yeast cells with SbbHLH85-BD. Three colonies were positive for X-a-gal and Aurobasidin (AbA). Among these candidates, only the binding of SbPHF1 and SbbHLH85 was stable. SbPHF1 encodes a SEC12-like protein and is homologous to AtPHF1. As a chaperone of phosphate transporter PHT1, SbPHF1 helps PHT1 transport to plasma membrane. To con rm their interaction in yeast, SbPHF1-AD vector was co-transformed into Y2H competent state with SbbHLH85-BD vector. As shown in Figure  7B, the experimental group grew normally on the medium which was added with X-a-gal but lacked isoleucine and tryptophan (SD/-L/-T/X), and the colony turned blue. Similarly, the experimental group grew normally on the medium added with the X-a-gal and AbA but lacking isoleucine, tryptophan, histidine and adenine (SD/-L/-T/-H/-Ade/X/A) and the colony turned blue. This shows that SbbHLH85 has a strong interaction with SbPHF1 (Figure7B).
To determine whether the interactions also exist in plant cells, we then used a BiFC system to verify the above observation. The agrobacterium tumefaciens of SbPHF1-C-YFP and SbbHLH85-N-YFP were mixed and transformed into tobacco leaves. At the same time, the empty carrier was combined with each fusion structure and injected into tobacco leaves. After incubation for 2 days, YFP signal was observed with a uorescence microscope. The co-transformed samples showed YFP uorescence in the nucleus, while none of the control samples showed any YFP signal ( Figure 7C). This demonstrates that SbbHLH85 and SbPHF1 were co-localized and interacted in plant nucleus.

Discussion
How plants nd a balance between environmental stress and plant growth is a new and important research topic (Monlau et al., 2015). A large number of indirect evidences have con rmed that the bHLH proteins are involved in the process of salt tolerance in plants (Babitha et  With the development of molecular biology, more and more bHLH transcription factors have been found, especially the identi cation of new atypical bHLH transcription factors, which makes this family more diverse. By analyzing the protein sequence of SbbHLH85 and bHLH transcription factors in other species, we found that RSL2 and RSL4 is the most closely related gene in Arabidopsis. But different from RSL2 and RSL4, SbbHLH85 has a Q5-A9-R13 motif as shown in Supplemental Figure1, indicating that SbbHLH85 is a new atypical bHLH transcription factor. On the one hand, it enriches the diversity of bHLH family in sweet sorghum. On the other hand, it indicates that SbbHLH85 may play a unique role in promoting salt tolerance and growth of sweet sorghum, which may be of great signi cance for crop improvement. bHLH transcription factors also affect the growth of root hairs. The Arabidopsis bHLH gene family members AtRHD6 and AtRSL1 regulate the development of root hair cells downstream of the transcriptional regulation complex that determines the fate of epidermal cells. A single mutant of the AtHRD6 gene has a small amount of root hairs; a single mutant of the AtRSL1 gene has normal root hairs, while the double mutants of these two genes have no root hairs (Menand et al., 2007). RSL4/RSL2 is related to the development of root hairs, and their expression is also regulated by both exogenous hormones and environmental signals. Auxin can activate RSL4 expression and control ROS related genes, including four peroxidases (PER1, PER44, PER73) (Mangano et al., 2017). Peroxidase genes (PERs) encodes class III peroxidase, and its activity is related to root hair elongation and ROS clearance (Kwon et al., 2015;Wanapu and Shinmyo, 1996). Similarly, RSL2 can inhibit the growth promoting effect of auxin by inhibiting ROS produced by peroxides (PERs) (Mangano et al., 2017). In this study, the expression of ABA and auxin pathway genes, peroxidase, receptor like protein kinase and root hair development related genes were changed by SbbHLH85 overexpression under salt stress ( Figure 6F-H). In conclusion, SbbHLH85 participates in ABA and auxin signaling pathways, controls the development of root hair and affects the content of ROS.
SbbHLH85 negatively regulates plant salt resistance by affecting plant nutrient distribution Under salt stress, the uptake of K + by root cells decreases and the absorption of Na + increases, which leads to the imbalance of ion homeostasis (Song et al., 2020). Recent studies have shown that plants can protect themselves by reducing the length and density of root hair and reducing the absorption area of excessive Na + when they sense stress signals (Lv et al., 2018;Wang et al., 2008).
Salt stress also can cause imbalance of nutrient distribution in plants, including phosphorus (P) (Yang et al., 2003). P is a key element of many biomolecules (nucleic acids, ATP, phospholipids) in many metabolic pathways; it is one of the important nutrients needed for plant growth and development (Marschner et al., 2005). Plant phosphorus uptake and transport through phosphate transporters, and phosphate transporters encoded by the phosphate transporter family (PHT1) genes are important proteins for plant acquisition and transport of phosphates (Shin et al., 2004). PHT1;5 is strongly induced in the root, leading to Pi mobilization changes between plant roots/grounds. Compared to the WT, PHT1;5 overexpression enhances root hair formation (Nagarajan, 2010).
The transport of PHT1 protein to plasma membrane is regulated by plant speci c companion protein PHF1. PHF1 gene encodes a plant speci c protein related to SEC12 protein structure, which locates in endoplasmic reticulum, and is strongly expressed in plant root cuticle, root hair and its cortical cells (Bayle et al., 2011). In 2005, a mutant of PHF1 was isolated in Arabidopsis. Studies on the mutant showed that the mutation of PHF1 reduced the accumulation of PHT1;1 transporter in plasma membrane, destroyed the transport of Pi and reduced the accumulation of Pi. In this study, SbbHLH85 is proved to interact strongly with SbPHF1. Therefore, we believe that the interaction between SbbHLH85 and SbPHF1 can destroy the transportation and accumulation of Pi in plants under salt stress, and aggravate the uneven distribution of nutrient elements, especially phosphorus, caused by high salinity, which greatly reduces the salt resistance of sweet sorghum.
In conclusion, SbbHLH85, different from RSL2 and RSL4 in Arabidopsis, is a necessary gene for root development in sweet sorghum. Knockout of SbbHLH85 in sorghum by CRISPR-Cas9 results in the inhibition of root development. We then show the molecular mechanism of SbbHLH85 regulating the salt response of sorghum by directly interacting with the speci c companion protein PHF1, which can affect the distribution of Pi. SbbHLH85 participates in the ABA and auxin pathways-mediated root hair development by affecting the expression of ABA and auxin pathway genes. The increase in the root hair number and length can promote the absorption of Na + . The increased Na + absorption and the decreased Pi content can ultimately result in the salt sensitive phenotype of sweet sorghum ( Figure 7D). In the future, it will be an effective measure to improve the salt tolerance of crops through properly reducing the number and length of root hairs upon presence of the stress.   Means with different letters are signi cantly different at P < 0.05.